CN101495865B - Rapid immunity chromatography detection of mouth cavity fluid - Google Patents
Rapid immunity chromatography detection of mouth cavity fluid Download PDFInfo
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- CN101495865B CN101495865B CN200580049809.2A CN200580049809A CN101495865B CN 101495865 B CN101495865 B CN 101495865B CN 200580049809 A CN200580049809 A CN 200580049809A CN 101495865 B CN101495865 B CN 101495865B
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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Abstract
The present invention relates to an oral fluid rapid immunochromatography test. More particularly, the present invention relates to an oral fluid collection swab separate from a lateral flow immunochromatography strip for detecting an analyte in oral fluid, consisting essentially of a sample pad, a conjugate pad, a test zone and control zone pad made of at least one matrix material, wherein the conjugate pad lies downstream of the sample pad, and is striped with a conjugate; the test and control zone pad lies downstream of the conjugate pad, wherein the test zone is immobilized with an specific binding reagent that specifically binding to the target analyte; and the control zone, downstream of the test zone, is immobilized with a second capture reagent. The invention also relates to a method for manufacturing the strip, a lateral flow immunochromatography method for detecting an analyte in oral fluid by using the strip, and kits containing the strip.
Description
Technical field
The present invention relates to mouth cavity fluid fast immune chromatographic to detect.More specifically, the present invention relates to for detecting in mouth cavity fluid the lateral flow immunochromatography inspection test-strips analyzing thing, collect the method for mouth cavity fluid sample, producing the method for described examination bar, analyzing the lateral flow immunochromatography method of thing and the kit containing this examination bar by using in this examination bar detection mouth cavity fluid.
Background technology
Be developed the analytical approach of various analysis thing in many qualitative or quantitative detection bio-tissues and fluid.Most current diagnosis detection adopts blood, urine, ight soil, biopsy or mouth cavity fluid.Compared with other material, collection mouth cavity fluid saliva and/or oral mucosa exudate bring relatively few privacy violation for detecting, and it is comparatively safe, and can relatively easily complete fast.
So far, for exploitation be used for the collection of mouth cavity fluid, transmission and sample preparation test-strips and for exploitation is based on the analysis of mouth cavity fluid, especially to the analysis of various antibody and metabolin, people have paid a lot of effort.
Such as, WO 88/07680 discloses and detects the existence of specific IgM, IgG and IgA in human or animal's body fluid (being selected from saliva, tears, seminal fluid, urine and cerebrospinal fluid) or the method for its amount by the radio immunoassay (RIA), enzyme-linked immunosorbent assay (ELISA) etc. of complexity are qualitative or quantitative, described method is difficult to carry out relatively, time-consuming and cost many.
U.S. Patent No. 5,103,836 disclose a kind of from the method for oral collection material for detecting, and it comprises the following steps: the absorption pad soaked into hypertonic salt solution is inserted oral cavity, and wherein the concentration of the salt of hypertonic solution in pad can reclaim the described material of high concentration effectively from oral cavity; Described pad is removed from oral cavity; Preserve this pad to take out collected material subsequently for ELISA from pad.Disclosed in disclosure file, absorption pad needs with hypertonic solution pre-service to provide the analysis thing of high concentration, and the ELISA reusing complexity carrys out detect analytes.
U.S. Patent No. 6,303,081 disclose for from oral collection and transhipment aqueous fluids to for detecting lateral chromatography examination bar inspection test-strips.Lateral chromatography examination bar to be placed in outer cover (housing) and to stretch along the chamber that outer cover limits.There is provided at least one to inspect (inspection) position to described lateral chromatography examination bar and inspect test result to realize selected position on lateral chromatography examination bar.Multi-hole center bar (wick) material at one end reaches the collection position of outer cover outside from described outer cover, try bar communicate at the other end and described lateral chromatography.Described multi-hole center strip material has particulate configurations, and described particulate absorption water-based mouth cavity fluid there is no absorption so that this fluid is delivered to described lateral chromatography examination bar from oral instructions.Mouth cavity fluid is easily discharged into lateral chromatography examination bar by this multi-hole center strip material.Due to the tortuous flow passage of this multi-hole center strip material, naturally prevent the reversed flow from lateral chromatography examination bar to oral cavity.Biteplate (bite plate) is connected with described outer cover and can inserts between patient teeth to be positioned by multi-hole center bar to collect mouth cavity fluid in oral cavity.Described biteplate is maintained at appropriate location to be positioned in oral cavity space by multi-hole center bar by dental articulation power (occlusal force) usually.When inspection test-strips is in mouth, obtain testing result rapidly by observing lateral chromatography examination bar.All disclose in U.S. Patent application No.2002/0192839, U.S. Patent application No.2002/0155029 and collect mouth cavity fluid to detect and/or quantitatively to analyze in mouth cavity fluid inspection test-strips and the method for the improvement of thing for a step, the content of these patents or application all with its entirety by reference to being incorporated to herein.
Although had directly by the analytic approach of above-mentioned inspection test-strips, fast and do not need the advantage of complex steps, for detected object, it be inconvenient for all will being engaged inspection test-strips in whole testing process.In addition, for the analysis with engineering noise instruction, when null result needs again to detect, or need the diagnosis carrying out making a collection of specimens confirming to detect to detect for the such routine of such as HIV, object will have to persist in for detection and be engaged inspection test-strips.But, should be understood that, one hour is reached after first collection, the collection of oral mucosa exudate reduces the available exudate being rich in IgG of oral tissue surfaces usually, it is convenient not that this will make to collect to use " one-stop sample divider/detection combination " again to detect subsequently, because detected object must wait until that exudate level is higher, otherwise carry out detection risk with sample and may have lower sensitivity (R.Mink, non-Publishing Data).
Summary of the invention
The present invention keeps in mind the above-mentioned shortcoming existed in prior art and makes, an object of the present invention is to provide a kind of simple, convenient and mouth cavity fluid collection method of cost-efficient side direction immunity-chromatography test strip, it is suitable for sensitive and analyzes thing in detection mouth cavity fluid rapidly.
Another object of the present invention is to provide a kind of method of producing described lateral flow immunochromatography inspection test-strips.
Another object of the present invention is to provide and is a kind ofly suitable for sensitive and detects in mouth cavity fluid the sensitive method analyzing thing rapidly.
Another object of the present invention is to provide for sensitive and detect in mouth cavity fluid the kit analyzing thing rapidly.
Therefore, in first, the invention provides a kind of for detecting in mouth cavity fluid the lateral flow immunochromatography inspection test-strips analyzing thing, it is substantially by sample pad, conjugate pad, detection zone and check plot pad composition, described pad is made up of at least one host material, wherein said conjugate pad is positioned at the downstream of sample pad and it draws striped with conjugate, described detection zone and check plot pad are positioned at the downstream of this conjugate pad and comprise detection zone and check plot, wherein detection zone is fixed with specificity combinating reagent, described specificity combinating reagent is combined with target analyte specifically, and check plot is fixed with the second capture agent.
In an embodiment in this regard, analysis thing to be detected is selected from the antibody of anti-following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
In another embodiment, described host material is selected from inorganic powder, such as silicon dioxide and aluminium oxide; Glass fiber filter paper; Natural polymer is especially based on cellulosic material, chromatographic paper; Natural polymer such as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, cross-link dextran, the agarose of synthesis or modification; And its combination.
In another embodiment, the host material of sample pad is glass fiber filter paper; The host material of conjugate pad is polyester material; The host material of detecting pad and contrast pad is nitrocellulose filter.
In another embodiment, conjugate comprises the label with the first capture agent coupling, and described first capture agent catches endogenous antibody in mouth cavity fluid.Described label is selected from colloid gold particle; Element or metallic sol particles, comprise selenium, silver, iron or carbon; Other Beads, comprises colored latex, liposome and dye granule.Preferably, described label is colloid gold particle.
In another embodiment, described first capture agent is selected from: the antibody of anti-igg, IgM or IgA, albumin A, Protein G and concanavalin A.Preferably, described first capture agent is albumin A.
In another embodiment, described specificity combinating reagent is selected from following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
In another embodiment, the antigen of communicable disease is the restructuring or the synthetic peptide that represent HIV protein immunization determining area, and described HIV albumen is especially selected from the HIV envelope protein of gp120 and gp41 of HIV-1 and the gp36 of HIV-2.
In another embodiment, the second capture agent is selected from: the antibody of anti-igg, IgM or IgA, albumin A, Protein G and concanavalin A, the antibody of wherein said anti-igg is goat anti-human IgG antibodies.
In second, the invention provides the method for the lateral flow immunochromatography inspection test-strips defined any one of production claim 1-17, it comprises: a) conjugate is drawn striped on conjugate pad; B) described specificity combinating reagent is fixed on the detection zone of detection zone and check plot pad; C) the second capture agent is fixed on the check plot of detection zone and check plot pad; D) with each pad described in blocking agent; And e) align gained pad make each other fluid be communicated with.
In one embodiment, drawing before striped, conjugate is stabilized in simple or complex sugar solution, and wherein said sugar juice comprises sucrose, trehalose, potassium stannate and carbamide peroxide.
In another embodiment, biotin/strepavidin linker is used to be fixed on detection zone and check plot by described specificity combinating reagent.
In another embodiment, biotin/strepavidin linker is used to be fixed on check plot by described specificity combinating reagent.
In another embodiment, described sealer comprises the detergent of nonionic, cationic, negative ion and amphoteric ion type; Sugar, comprises sucrose, fructose; Or protein, comprise that bovine serum albumin(BSA), animal's whole blood are clear, casein and skimmed milk power.
In another embodiment, animal's whole blood is clearly hyclone.In another embodiment, animal's whole blood is clearly for being selected from the avian serum of goose serum, turkey serum and chicken serum.
In the 3rd, the invention provides for detecting in mouth cavity fluid the lateral flow immunochromatography method analyzing thing, it comprises: (a) uses and the lateral flow immunochromatography defined any one of claim 1-17 is tested and tried the gatherer that bar separates and collect mouth cavity fluid; B gatherer drops in the sample buffer of certain volume and obtains potpourri to be discharged in damping fluid by mouth cavity fluid by (); C the lateral flow immunochromatography defined any one of aforementioned claim test examination bar is put into described potpourri by (); (d) by observing the existence of check plot signal to determine the validity detected, and by detect start after observe signal in detection zone in 15-60 minute existence determine to analyze the existence of thing.
In another embodiment, the gatherer in step (a) is untreated polyester swab (swab), such as Texwipe Large Alpha Swab TX714A.
In another embodiment, sample buffer is the buffer solution of potassium phosphate of pH7.2+/-0.2, and it also comprises 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween 80,0.2%Tetronic T-904 and 0.0.285% (active component) ProClin 950.
In another embodiment, the volume of sample buffer is 1000 μ l.
In another embodiment, described method is also included in the step of taking out potpourri sample between step (b) and step (c), and the volume of wherein said sample is 200 μ l.
In another embodiment, described signal for there being colo(u)r streak, as blush (reddish) line.
In another embodiment, in 20-45 minute, described signal is observed.
In the 4th, the invention provides for detecting in mouth cavity fluid the kit analyzing thing, it comprises at least one above-mentioned lateral flow immunochromatography inspection test-strips.Described inspection test-strips is closed in drying receptacle.Each kit optionally comprises sample buffer, mouth cavity fluid gatherer, provides the package insert of appended examination bar instruction, tubule (vial) containing the positive and negative control for carrying out quality testing to inspection test-strips, can be used for determining that the present invention measures the timer and/or bio-hazard thing container handling when completed.
In a preferred embodiment of this aspect, sample buffer is the buffer solution of potassium phosphate of pH7.2+/-0.2, and it also comprises 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween 80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin 950.
In another embodiment, gatherer is untreated polyester swab, such as TexwipeLarge Alpha Swab TX714A.
Therefore, an advantage of the invention is the simplicity of design and the low cost of material, this is owing to lacking outer cover, such as this is found in other mouth cavity fluid and detects (for example, see United States Patent (USP) 6303081, U.S. Patent application 20020155029 and 20020192386) and other quick lateral flow detection (United States Patent (USP) 6,303,081,5 fast, 935,864,6,485,982,6,534,320,6,352,862,6,187,598 and 6,027,943) in.This not only makes the cost of product manufacturing lower, but also provides the manufacturing process complicacy of more low-complexity, This further reduces cost.
Another advantage of the present invention is that this analysis self is independent of sample divider (such as in United States Patent (USP) 6303081, U.S. Patent application 20020155029 and 20020192839 and United States Patent (USP) 5935864), and this makes user carry out repeated detection when not needing collection in addition to sample.This is especially favourable in the lateral flow immunochromatography with engineering noise instruction measures, wherein invalid result needs again to detect, or need to carry out confirming that the diagnosis of detection detects to collected sample to be also especially favourable for such as HIV is conventional like this, because reach one hour after first collection, oral mucosa exudate collect usually reduce oral tissue surfaces can the effluent being rich in IgG, it is convenient not to make to collect subsequently again to detect with " one-stop sample divider/detections is combined ".
The further advantage of the present invention is to detect (such as United States Patent (USP) 6 with other mouth cavity fluid, 303,081) compare, closely (such as 5mm × 1mm × 75mm), this makes transport and storage cost reduce to described mouth cavity fluid fast immune chromatographic examination bar.
Another advantage of the present invention is that described mouth cavity fluid collection method provides and enough detects for other volume judging that such as authenticity detects.
The further advantage of the present invention is to detect (such as United States Patent (USP) 6 compared to other mouth cavity fluid, 303,081) complicacy of producing reduces, because need not configure outer cover around lateral flow chromatography examination bar, this causes material cost and process time to be all minimized.
Accompanying drawing explanation
Fig. 1 illustrates the program detected according to the mouth cavity fluid fast immune chromatographic of a preferred embodiment of the present invention, and wherein Fig. 1-1 to 1-15 illustrates the step of detection.
Fig. 2 illustrates according to a preferred embodiment of the present invention, three kinds of possible outcomes that mouth cavity fluid fast immune chromatographic detects, and wherein Fig. 2 A, 2B and 2C represents feminine gender, the positive and invalid result respectively.
The description of preferred embodiment
By to following detailed description of the preferred embodiments also by reference to the accompanying drawings, the present invention can be understood better.
In first, the invention provides a kind of for detecting in mouth cavity fluid the lateral flow immunochromatography inspection test-strips analyzing thing, it is made up of sample pad, conjugate pad, detection zone and check plot pad substantially, described pad is made up of at least one host material, wherein conjugate pad is positioned at the downstream of sample area pad, and draw striped thereon with conjugate, described detection zone and check plot pad are positioned at the downstream of this conjugate pad, and be fixed with the specificity combinating reagent with target analyte specific binding in detection zone, be fixed with the second capture agent in check plot.
Described sample pad receives oral cavity fluid sample.Sample pad is made up of the material showing low target antibody reservation usually.In some embodiments, sample pad also can play the effect of mechanical filter, and it can catch any unwanted particle such as dust, fragment, precipitation, mucus or blood.
Described conjugate pad is positioned at the downstream of sample pad and containing conjugate, described conjugate comprises directly or indirectly and the label of the first capture agent coupling.
Described detection zone is positioned at the downstream of conjugate pad and containing specificity combinating reagent, described specificity combinating reagent is combined with target antibody specifically, and whereby, the conjugate of mark can be fixed in matrix.
Described check plot is arranged in the downstream of stream detection zone.The second capture agent is contained in check plot, and described second capture agent catches the antibody of being caught by the first capture agent, and is preferably fixed on to form control line in check plot, and described control line concentrates any by the labeled antibody conjugate of the second capture reagent bind.
As used herein, term " mouth cavity fluid " refers to be found in one or more fluids intraoral alone or in combination.It includes but not limited to saliva and oral mucosa exudate.It will be appreciated that, mouth cavity fluid can comprise from multiple source (the such as attached body of gland parotid gland, salivary gland, sublingual gland, gingival mucosa and oral mucosa) the combination of fluid, and term mouth cavity fluid comprises the fluids from each these source alone or in combination.Term saliva refers to the combination of mouth cavity fluid, such as, usually see oral cavity, especially after chewing.As used herein, term " oral mucosa exudate " refers to the fluid that produced from oral mucosa space to Passive diffusion oral cavity by serum component.Oral mucosa exudate usually forms a kind of component of saliva.
As used herein, term " analysis thing " is used in reference to part to be detected in particular analysis.Usually the analysis thing detected in the present invention analyzes includes but not limited to the antibody of anti-following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.Particularly preferred analysis thing comprises the antibody of the antigen of infectivity resistant disease, described communicable disease such as HIV, hepatitis etc.Antigen can be but be not limited to such as hepatitis B antigen, and antibody can be but be not limited to the antibody of the antibody of HIV, the antibody of HTLV, the antibody of helicobacter pylori, the antibody of hepatitis, the antibody of measles, parotitic antibody and rubella.The metabolic product of medicine and drug abuse or drug abuse can be but be not limited to tetrahydrocannabinol, nicotine, ethanol, theophylline, phenytoinum naticum, paracetamol, lithium, stable, nortriptyline (nortryptyline), quinalbarbitone, phenobarbital.Hormone can be but be not limited to testosterone, estradiol, 17-hydroxyprogesterone, progesterone, thyroxine, thyrotropic hormone, follicle-stimulating hormone (FSH) and luteinizing principle.
As used herein, " specificity combinating reagent " is the reagent with target analyte specific binding, as mentioned above, described target analyte is selected from following antigen: the metabolic product of communicable disease, hormone, growth factor, medicine, drug abuse and drug abuse.
As used herein, " antigen " is a kind of material stimulating antibody tormation when it is introduced in mammal or birds.The preferred antigen of the present invention comprises human immunodeficiency virus (HIV) protein, particularly gp120 and gp41 of virus envelope protein HIV-1 and the gp36 of HIV-2.
As used herein, " antibody " refers to the protein that is made up of one or more polypeptide of substantially being encoded by immunoglobulin gene or immunoglobulin gene fragment.The immunoglobulin gene of generally acknowledging comprises κ, λ, α, γ, δ, ε and μ constant region gene, and the immune globulin variable region gene of huge amount.The light chain of antibody is divided into κ or λ.Heavy chain is divided into γ, μ, α, δ or ε, itself so that determine that the type of immunoglobulin (Ig) is respectively IgG, IgM, IgA, IgD and IgE.
The basic structural unit of known antibodies (also claiming immunoglobulin (Ig)) comprises the tetramer.Each tetramer forms identical polypeptied chain by two, and often pair has a light chain (about 25kD) and a heavy chain (about 50-70kD).The N of each chain holds about 100 to 110 of a definition primary responsibility antigen recognizing or more amino acid whose variable region.Term variable region light chain (V
l) and Variable region heavy (V
h) refer to these light chains and heavy chain respectively.
Antibody can be used as complete immunoglobulin (Ig) or exists as the fragment passing through a large amount of well-characterized produced with various peptidase digestion.Therefore, such as pepsin region produces F (ab ')
2, F (ab ')
2be the dimer of Fab, Fab is from as by disulfide bond and V
h-V
h1 light chain be connected.F (ab ')
2the disulfide bond that can be reduced to destroy hinge region in a mild condition connects thus by F (ab ')
2dimer is converted into Fab ' monomer.Fab ' monomer is the Fab (about the more detailed description of other antibody fragment see Fundamental Immunology, W.E.Paul, Ed., Raven Press, N.Y., 1993) with a hinge area part substantially.Although various antibody fragment defines according to the digestion of complete antibody, technician will know such Fab ' fragment by chemistry or by utilizing recombinant DNA method de novo formation.Therefore, term antibody also comprises antibody fragment as used herein, and described antibody fragment produces by modifying complete antibody or uses recombinant DNA method de novo formation.
As used herein, term " matrix " refers to insoluble material that fluid can be supported to flow.That host material can be natural origin and/or synthesis source, (bibulous) of water suction or do not absorb water, fibrous or granular.Matrix of the present invention can be formed as the continuous strip of same material or different materials potpourri, and described different materials is along common band continuous distribution, or discontinuously arranged to be formed in the zone that band zones of different has different physics or chemical property.As an alternative, the pad of series of discrete can be formed by identical or different host material, and the reagent for measuring is added to each pad.Then pad can be placed to fluid communication with each other to form continuous print stream.Material for building matrix of the present invention can be inertia or can with one or more reagent reactings of the present invention, it is insoluble that to be described material be still prerequisite in enforcement invention described herein.Host material can be selected from inorganic powder, such as silicon dioxide and aluminium oxide; Glass fiber filter paper; Natural polymer is particularly based on cellulosic material such as filter paper, chromatographic paper; Natural polymer such as cellulose nitrate, cellulose acetate, Polyvinylchloride, polyacrylamide, cross-link dextran, the agarose of synthesis or modification; And its combination.In a preferred embodiment, described matrix is nitrocellulose filter.
In another preferred embodiment of the present invention, glass fiber filter paper is used as the matrix of sample pad, and polyester material is used as the matrix of conjugate pad, and nitrocellulose filter is used as the matrix of detection zone and check plot pad.
" water suction " (bibulous) refers to that some absorbent material supports the ability of the different solute migration speed of otherness during fluid flows through this material.Therefore, solute is carried out chromatography by absorbent material energy physically based deformation and/or the chemical property with water suction character.
As used herein, term " label " refers to directly or indirectly through analyzing thing specific binding partner and detectable atom or the molecular structure of analyzing thing specific binding.Label of the present invention can by physics or chemical detection.Preferred label is visible to bore hole when joined, to indicate positive test symbol.Label of the present invention is selected from colloid gold particle; Element or metallic sol particles, comprise selenium, silver, iron or carbon; Other Beads, comprises colored latex, liposome and dye granule; In the present invention's preferred embodiment, described label is colloid gold particle.These labels are well known in the art, and are described in such as G.FrensNature, 241,20-22 (1973) and U.S. Patent No. 4,313, in 734, its disclosure with its entirety by reference in being incorporated herein.
" collaurum " used in this invention refers to the colloidal sol of fine gold grain, and it can ad infinitum be held in aqueous suspension.
As used herein, " capture agent " is any molecule being attached to target antibody specifically.Capture agent of the present invention is preferably fixed on matrix with deterministic model, typically is the line vertical with stream.Preferred capture agent is the antibody of anti-igg, IgM or IgA; Albumin A, Protein G or concanavalin A.
As used herein, " the first capture agent " of the present invention is the capture agent with label coupling.Preferably, described first capture agent is the antibody of anti-igg, IgM or IgA; Albumin A, Protein G or concanavalin A.In a preferred embodiment, albumin A or Protein G and label coupling, form conjugate.
" albumin A " used in this invention refers to the high stability surface receptor produced by staphylococcus aureus (Staphylococcusaureus), its (Boyle that can combine with the Fc part of the immunoglobulin (Ig) of a large amount of species especially IgG, M.D.P.and K.J.Reis.Bacterial FcReceptors.Biotechnology 5:697-703,1987).A Protein A molecules can combine at least 2 IgG molecule (Sj simultaneously
quist, J., Meloun, B.and Hjelm, H.Protein Ais isolated from Staphylococcus aureus after digestion with lysostaphin.Eur J Biochem 29:572-578,1972).
" Protein G " used in this invention refers to from the streptococcic cell surface associated proteins be combined with IgG with high-affinity.It has the IgG binding structural domain (see Lian, et al., 1992.Journal of Mol.Biol.228:1219-1234 and Derrick andWigley.1994.Journal of Mol.Biol.243:906-918) of three very high homology.
As used herein, " the second capture agent " be fixed on check plot with catch by the first capture agent the capture agent of antibody of catching.Suitable the present invention second capture agent is anti-igg, the antibody of IgM or IgA, albumin A, Protein G or concanavalin A.In a preferred embodiment, described second capture agent comprises the anti-igg antibody from being different from the species providing mouth cavity fluid.Even more preferably, this second capture agent is anti-human IgG antibodies; Most preferably, this second capture agent is goat anti-human IgG antibodies.
As used herein, " stream " refers to when oral cavity fluid sample is through the route of matrix institute warp.Described stream is preferably uniline, but can comprise several routes, and wherein every bar route can simultaneously, sequentially or independently support liquid flow for other route.
" downstream " refers to that liquid is by the directed stream away from liquid application point during matrix.
" upstream " refer to liquid by during matrix towards the directed stream of liquid application point.
In second, the invention provides the method for producing lateral flow immunochromatography inspection test-strips, it comprises: a) conjugate is drawn striped on conjugate pad; B) specificity combinating reagent is fixed to the detection zone of detection zone and check plot pad and the second capture agent is fixed to check plot; C) with each pad of blocking agent; With the gained pad that e) aligns, fluid is each other communicated with.
conjugate is drawn striped on conjugate pad
Conjugate is deposited in the matrix of conjugate pad in the following manner: when itself and mouth cavity fluid sample contacts, and it is easy to migration in fluid stream.For realizing this point, the matrix of conjugate pad is made up of the polyester of adhesive network (spun-bonded).Such as contact tip or gasoloid tip is utilized by conjugate to draw striped together with stroke striped solution on padding.Before drawing striped, preferably by conjugate stabilization.Such as, can by conjugate stabilization in 20% sucrose, 5% trehalose and 0.1% carbamide peroxide.
When oral cavity fluid sample flows through conjugate pad, conjugate is dissolved and is added with the fluid stream of inspection test-strips.
specificity combinating reagent is fixed to detection zone
Use the methods known to those skilled in the art, be applicable to specificity combinating reagent of the present invention and can comprise natural, chemosynthesis or recombinant production from any source and obtain.Such as, the peptide moiety of preferred SEQ ID No.1 to 5 can use the chemosynthesis of Solid phase peptide synthesis technology, or coding is expected the nucleic acid of peptide to be connected in expression vector and in suitable host, to express described nucleic acid and carry out recombinant production by operability.Once be separated, known technology can be used described peptide biotinylation.
Can use any the methods known to those skilled in the art that suitable antigen is fixed to matrix, described method does not destroy the specific binding of antigen to target antibody.Preferably, for recombinant protein antigen, antigen is fixed directly in matrix without modifying further.Preferably, use biotin/strepavidin linker antigenic synthetic peptide is fixed in matrix, most preferably, antigen is coupled to biotin and with streptavidin compound, then streptavidin is coupled to matrix.For water absorptivity matrix, use and well known to a person skilled in the art technology, usually before closing, the streptavidin of compound is coupled to matrix.Preferably, contrast in the solution of each biotin structure at the streptavidin binding site equivalent containing at least 4: 1 ratio and realize coupling, although in other ratio such as 0.5: 1,1: 1,2: 1,3: 1 and 5: 1 and all centres (mark) ratio are also included within, as a part of the present invention.For water absorptivity matrix, resulting composite can be applied to host material and drying simply, closes subsequently with suitable sealer.There is enough high molecular and the suitable antigen that can directly be combined with matrix such as a lot of recombinant protein need not be adhered to by the method for biotin/strepavidin linker.The amino acid sequence being applicable to Exemplary antigens peptide of the present invention sees SEQ ID No:1 to 5.
Antigen is fixed to matrix preferably to carry out for the mode of concentrated labeled antibody conjugate, described antibody coupling matter is specifically in conjunction with immobilized antigen.By the antibody coupling matter of concentrated mark, the signal produced by label is reinforced, thus improves the possibility that sensitivity also reduces to obtain error result as far as possible.
second capture agent is fixed on check plot
The second capture agent is contained in check plot, and is preferably fixed in check plot to form the control line of concentrated label conjugate.Adopt known technology, comprise the above-mentioned technology for immobilized antigen, the second capture agent of the present invention will be applicable to and be fixed to matrix.Can use biotin/strepavidin linker that the second capture agent is fixed to matrix, in the case, most preferably, as mentioned above, before streptavidin is coupled to matrix, the second capture agent be coupled to biotin and meet with it with streptavidin.Preferably, capture agent such as Goat anti human IgG F (ab ')
2biotin/strepavidin linker can not be used and be fixed directly in matrix.
by pad blocking agent
Although intrinsic absorptive host material can be used to make pad of the present invention, flow through fluid flowing that the present invention examines test-strips preferably in itself for unwetted.
By application sealer, water-absorbing material can be converted into the material showing unwetted flow characteristics.These sealers can be detergent, sugar or protein, and it can hinder the interaction force producing water absorption character.Exemplary protein sealer comprises bovine serum albumin(BSA) self or it methylates or succinylation form, animal's whole blood clear (such as horse or hyclone), and other blood protein.Preferred sealer is avian serum such as goose or turkey serum, most preferably chicken serum.Other example of protein blocking agent comprises casein and skimmed milk power.Sealer based on detergent is selected from nonionic, cationic, anionic and amphoteric ion type, and it selects to be the character based on being closed matrix.Tween 20 is the useful especially detergents for closing membrane.The exemplary sugar that can be used as sealer comprises sucrose and fructose.
Sealer is applied by padding to remove undesirable surface reaction described in the capping agent solutions process by effective concentration.Generally speaking, this process adopts lock solution to carry out several minutes at about room temperatures to a few hours, the protein solution of described lock solution such as 1-20mg/ml protein.Then gained coating material is for good and all adsorbed onto surface by air oxygen detrition, freeze-drying or other drying means.
Use intrinsic water absorptivity but the matrix that can change unwetted flow characteristics into is particularly useful for fixing specificity combinating reagent and the second capture agent.Such as, can before applying sealer, the second capture agent be applied to matrix and can fix in position.Once the second capture agent is fixed to matrix, then sealer can be applied.
Such as, during operation the present invention, by sealer of the present invention to be enough to stop the amount of target antibody and host material interphase interaction to be applied to sample pad.The particularly advantageous sealer of one for sample pad is avian serum, more preferably chicken serum.In a preferred embodiment, sample pad is with containing the solution saturates of following composition: polyvinylpyrrolidone, bovine serum albumin(BSA), avian serum, borate and/or carbonate buffer solution (about 0.5M) and Triton X-100 or Tween-20 detergent.To the sample pad be made up of glass fiber material, most preferred sample Block buffer is made up of following: 40% chicken serum (hot deactivation aseptic filtration), 0.25M saleratus, 0.05M dipotassium hydrogen phosphate, 0.1%Tween 80,100mM potassium stannate and 0.2% carbamide peroxide, its pH is 8.2 to 8.5.By pad compression to remove excessive damping fluid and will pad 30 DEG C of dried overnight.An advantage of the method is wettable and the capillary action increase of sample pad.
The composition of Block buffer and pH change with the material type of pad.Such as, closed by the conjugate be made up of polyester material pad being immersed in the damping fluid containing following composition: polyvinylpyrrolidone, chicken serum, bovine serum albumin(BSA), 0.1% carbamide peroxide, 100mM potassium stannate and sal tartari and/or borate buffer.Then by conjugate pad dry 120 minutes of 50 DEG C and forced ventilation, then air-dry overnight under environment temperature.For the detection zone of being made up of nitrocellulose filter and check plot pad, most preferred sample Block buffer composition is: 0.15% bovine serum albumin(BSA), 0.075 Tween 20 in the potassium salt soln that pH7.8+/-0.1 is cushioned.
In the 3rd, the invention provides for detecting in mouth cavity fluid the lateral flow immunochromatography method analyzing thing, it comprises: (a) collects mouth cavity fluid with examining with lateral flow immunochromatography the gatherer that test-strips separates; B gatherer drops in the sample buffer of certain volume and obtains potpourri to be discharged in damping fluid by mouth cavity fluid by (); C lateral flow immunochromatography inspection test-strips as above is placed in potpourri about 15-60 minute by (); D () determines the validity detected by the existence observing check plot signal, and determine by the existence observing detection zone signal the existence analyzing thing.
The principle of said method sees following.Mouth cavity fluid is also upwards moved along measuring inspection test-strips by wicks by matrix.It is by conjugate rehydration, such as try erythroid protein A-colloidal gold reagent on bar, and the IgG in sample is combined with conjugate and forms IgG/ conjugate compound.Described IgG/ conjugate compound continues upwards to move along examination bar, and the detection zone first arrived containing specificity combinating reagent in inspection test-strips, wherein said specificity combinating reagent can be combined with target analyte specifically, and compound is fixed in detection zone and occurs signal, such as erythroid have colo(u)r streak.This Indicator Reaction or positive findings.Do not have signal designation sample not containing target analyte in detection zone and form non-reacted or negative findings.IgG/ conjugate compound continues upwards to move until it arrives check plot along measuring examination bar.The second capture agent is contained in check plot, such as, be fixed on the Goat anti human F (ab ') in the line measuring inspection test-strips
2igG fragment.Remaining IgG/ conjugate compound and immobilization F (ab ')
2fragment combines, and occurs signal, and such as erythroid have colo(u)r streak.The appearance of this signal is the normal work of test and contains the evidence of IgG.No matter sample is reactive or non-reacted for target analyte, is carrying out all having signal to occur in check plot between all effective detection periods.Sample continues through check plot migration and enters final absorption pad, and it helps pull IgG/ conjugate compound by examination bar and remove any background color.
In this regard, the sample divider that can be used for this test can from oral collection oral mucosa exudate, and known described oral mucosa exudate has the higher diagnostic IgG needed for other products (for example, see U.S. Patent No. 5,103,836).In fact, in a preferred embodiment, the gatherer for this mensuration is ready-made untreated polyester swab, i.e. Texwipe LargeAlpha Swab, TX714A (Texwipe Inc., Upper Saddle River NJ).
In an embodiment in this regard, be with the 0.15M sodium chloride of pH7.2+/-0.2 potassium phosphate buffering, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween 80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin 950 for diluting the sample buffer of mouth cavity fluid.1000 μ l for diluting the volume of the sample buffer of mouth cavity fluid.
In another preferred embodiment, the method is also included in the step of taking out potpourri sample (aliquot) between step (b) and step (c).In a specific embodiment, the volume of described sample is 200 μ l.
When proper operation, continuation is combined markd antibody coupling matter by the second capture agent, until unconjugated labeled antibody conjugate is consumed totally, or the second capture agent is saturated.Because even contain endogenous IgG from the mammiferous mouth cavity fluid sample of health, and with the molar weight of the labelled reagent of the label coupling molar weight preferably greater than immobilized antigen, the antibody coupling matter of mark should always can be used in conjunction with the second capture agent, produces signal at control line.Therefore, the signal designation that can't detect control line or the human IgG lacking q.s are to produce object line, or inspection test-strips defectiveness, or examination bar misoperation.
Typically, after described inspection test-strips inserts mouth cavity fluid, can be observed label signal, more preferably between 15 to 45 minutes, most preferably between 15 to 30 minutes between 15 to 60 minutes.Detection start after early than 15 minutes or be later than 60 minutes read testing result may to the result made mistake.The signal produced by colored labels as above directly can be detected from inspection test-strips without processing further usually.Fluorescent marker may need photofluorometer to detect.In method well-known to those skilled in the art, use silver salt solution can strengthen the signal produced by metal-sol label.Similarly, when using enzymes, label must with the substrate contact of enzyme marker, can product be detected to produce.Therefore, these Enhancement Method deviate from the conventional single stage method mensuration adopting coloured particle label and colloidal sol to carry out, because matrix must contact with imaging liquid (silver salt or substrate solution) before certification mark thing.
In a most preferred embodiment, the invention provides a kind of inspection test-strips, it contains recombinant protein or the synthetic peptide of the immune determining area representing HIV-1 gp41 and HIV-2 gp36 envelope protein, and Goat anti human IgG F (ab ')
2fragment antibody prize procedure contrasts, and they are respectively fixed on the nitrocellulose filter of detection zone and check plot.
Fig. 1 illustrates the program that mouth cavity fluid fast immune chromatographic according to a preferred embodiment of the invention detects, and wherein Fig. 1-1 to 1-15 illustrates the step of detection.For testing, with the gums up and down of polyester swab wiping object, then described swab be placed in test tube about 1000 μ l mouth cavity fluid sample buffer (the 0.15M sodium chloride of pH7.2+/-0.2 potassium phosphate buffering, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween 80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin 950) and mix at test tube.Extrude the fluid in swab and discard, 200 these sample mixture of μ l are transferred to clean tube to measure.Mensuration examination bar is put into the test tube containing 200 μ l sample mixture vertically.When the sample diluted upwards moves along mensuration examination bar, it makes blush protein A-colloidal gold reagent rehydration on examination bar, and in sample, IgG is combined with albumin A/colloid gold particle and forms IgG/ conjugate compound.Described IgG/ conjugate compound continues upwards to move along examination bar, first arrives the detection zone (the HIV antigen that can be combined with ANTI-HIV DRUGS is contained in described detection zone) that measures examination bar and the antigen line being fixed in detection zone occurs erythroidly having colo(u)r streak.This Indicator Reaction or positive findings.The intensity of line is not proportional with the antibody amount that exists in sample.Lack in detection zone and have colo(u)r streak indicateing arm originally not containing ANTI-HIV DRUGS.IgG/ conjugate compound continues upwards to move until it arrives check plot along measuring examination bar.Check plot contain be fixed on measure examination bar reach the standard grade in Goat anti human F (ab ')
2igG fragment antibody.Remaining IgG/ conjugate compound and immobilization F (ab ')
2fragment combines, and occurs erythroidly having colo(u)r streak.The appearance of control line is the evidence detecting normal operation and contain IgG.No matter the antibody of sample to HIV-1 or HIV-2 is reactive or non-reacted, all will occur in check plot carrying out blush control line between all effective detection periods.Sample continues to migrate through check plot and enters final absorption pad, and described absorption pad helps pull IgG/ conjugate compound by examination bar and remove any background color.Mensuration examination bar being introduced in dilution sample 20 minutes but after being no more than 45 minutes, make an explanation to result.Detection start after early than 20 minutes or be later than 45 minutes read testing result may to the result made mistake.Remaining dilute sample can be used for other and detects, such as authenticity test.
The result that Fig. 2 three kinds of illustrating that mouth cavity fluid fast immune chromatographic according to a preferred embodiment of the invention a detects are possible, wherein Fig. 2 A, 2B and 2C represents feminine gender, the positive and invalid result respectively.
Fig. 2 A shows non-reacted result, wherein only occurs single line in check plot, points out and lack active anti-HIV-1 or AntiHIV1 RT activity-2 antibody in mouth cavity fluid sample.This testing result is interpreted as HIV antibody feminine gender.
Fig. 2 B shows reactive result, and wherein detection line and control line all occur, namely in inspection test-strips, two lines appear at detection zone and check plot respectively.In these lines one comparable another is darker.Reactive result means and in mouth cavity fluid sample, anti-HIV-1/2 antibody detected.It is positive that this testing result is interpreted as preliminary HIV antibody.
Fig. 2 C shows null result, does not wherein have control line in check plot.Even if there is detection line in detection zone, result is also invalid.Invalid test should adopt new inspection test-strips to carry out repeated test.
Above-mentioned embodiment can design with alternative, and it comprises substituting label outside aurosol.Such as, other label includes but not limited to element or metal-sol such as selenium, silver, iron or carbon, other Beads such as colored latex, liposome and dyed particles.Energy specificity is caught other first capture agent of antibody in mouth cavity fluid sample and is included but not limited to the antibody of anti-IgM or IgA; Protein G, or concanavalin A.Second capture agent also can be mixed with and include but not limited to alternative part, such as albumin A or Protein G.
In the 4th, the invention provides the kit analyzing thing for detecting mouth cavity fluid, it comprises above-mentioned disposable lateral flow immunochromatography inspection test-strips.Inspection test-strips is closed in dry container.Each kit optionally comprises sample buffer, mouth cavity fluid gatherer, provides the package insert of appended inspection test-strips operation instruction, is equipped with for the tubule of the positive and negative control of inspection test-strips being carried out to quality inspection, the timer that can be used for determining when mensuration of the present invention completes and/or bio-hazard product container handling.
In a preferred embodiment in this regard, sample buffer is the solution of pH7.2+/-0.2 potassium phosphate buffering, and it is also containing 0.15M sodium chloride, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween 80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin 950.
Although for knowing and being convenient to understand, the mode of explanation and example describe in detail foregoing invention by way of example, but it is apparent to those skilled in the art, according to instruction of the present invention, can make a variety of changes it and revise and without prejudice to the spirit and scope of claims.
Except diagnosing except HIV by detecting HIV antibody in mouth cavity fluid, the present invention easily can be configured for the diagnosis of many illnesss, and these illnesss need to carry out immune detection to the analysis thing in mouth cavity fluid.Especially easily use concept of the present invention to be used for sexually transmitted disease to detect, such as syphilis antibody and hepatitis virus antibody.
Embodiment
Following examples will illustrate the present invention but not be limited.
embodiment 1: the production of immunochromatography inspection test-strips
Provide the inspection test-strips for the antibody test of quick HIV-1/2 mouth cavity fluid in the present embodiment, wherein use glass fiber material as the matrix of sample pad, use polyester material as the matrix of conjugate pad, use nitrocellulose filter as the matrix detected and contrast is padded.
The S & S S-33 glass fiber material Block buffer of 1 inch is soaked, described Block buffer is made up of following: 40% normal chicken serum (hot deactivation), 0.25M saleratus, 0.05M dipotassium hydrogen phosphate, 0.1%Tween 80,100mM potassium stannate and 0.2% carbamide peroxide, its pH is 8.2 to 8.5, under room temperature (15-30 DEG C) in low humidity room dry 8 hours, then spend the night in 50 DEG C in drying receptacle, and kept dry.
By using, gasoloid is most advanced and sophisticated draws striped by pAg conjugate on pad, prepares conjugate pad from polyester film.Before drawing striped, by conjugate stabilization in 20% sucrose, 5% trehalose, 100mM potassium stannate and 0.1% urea peroxide.Then described pad to be immersed in the damping fluid containing polyvinylpyrrolidone, chicken serum, bovine serum albumin(BSA) and carbonate buffer agent and to use forced drafts dry 50 minutes at 50 DEG C.
HIV antigen can be coupled to detection zone pad, and it comprises usage chain avidin/biotin connexon.In order to draw striped, trying bar with 300ng/ examination bar and 0.15ng/ respectively and the HIV-I peptide of synthesis and the HIV-2 peptide (such as SEQ ID No:1 to 5) of synthesis are put on detecting pad.The solution be made up of 1.2mg/mlHIV-1,0.06mg/ml HIV-2,4.36mg/ml affinity element and 0.05% isopropyl alcohol is used to draw striped on detecting pad.1mg/ml Goat anti human IgG Fc F (ab) 2 is put on contrast pad.
For using the inspection test-strips of Recombinant HIV-1/ Recombinant HIV-2,0.4-0.7 μ g/ in 0.001%Tween 80,5% sucrose and 2% methyl alcohol is tried the gp36 albumen that the gp41 albumen of bar and 0.04-0.08 μ g/ try bar and put on detection zone, and 0.175 μ g/ in 5% sucrose, 2% methyl alcohol and 0.01M sal tartari pH8.4 is tried the Goat anti human IgG F (ab ') of bar
2put on check plot.
By the detection zone that as above processes and check plot blocking agent, described sealer is made up of 0.15% bovine serum albumin(BSA) in the kaliumphosphate buffer of pH7.8+/-0.1,0.075%Tween 20.Then the alignment of gained pad is communicated with to make it mutual fluid, makes conjugate pad be positioned at the downstream of sample pad; Detection and check plot pad are positioned at the downstream of conjugate pad, with the downstream making check plot be positioned at detection zone.
embodiment 2: mouth cavity fluid fast immune chromatographic detects
The mouth cavity fluid fast immune chromatographic using the present invention to try bar is provided to detect, as shown in Fig. 1-1 to Fig. 1-15.First, biased sample damping fluid (with the 0.15M sodium chloride of pH7.2+/-0.2 potassium phosphate buffering, 0.1%Triton X-100,15% hot deactivation chicken serum, 30 μ g/ml affinity elements, 0.2%Tween 80,0.2%Tetronic T-904 and 0.0285% (active component) ProClin 950) is taken for about 3 times by inverted bottle lightly.Take off bottle cap (Fig. 1-1) from bottle and damping fluid be filled to dropper to groove (Fig. 1-2).The entire contents of dropper is dispensed to (Fig. 1-3) in test tube.Then one is taken off by wrapping the clean swab provided.Catch swab handle.Avoid the fabric end of touching swab.Subsequently, suitable pressure is applied also gently with gums line in swab fabric end to and fro wiping.One jiao at mouth starts softly and lentamente wiping until arrive another angle (Fig. 1-4) of mouth, then returns starting point (about 5-6 second) (Fig. 1-5) along the wiping of upper gums line.Then swab is rotated with gums (Fig. 1-6) under the opposite side wiping using swab.The opposite side using swab softly and lentamente gums line under wiping back and forth.One jiao at mouth starts (Fig. 1-7), terminates at another angle of mouth, then returns to place of setting out (about 5-6 second) (Fig. 1-8) along the wiping of lower gums line.Immediately swab is put in and fills (Fig. 1-9) in the pipe of sample buffer.
Hold swab handle.Swab back and forth to be inserted in sample buffer pipe 6-8 time, be close to the both sides (Fig. 1-10) of pipe friction swab.Swab (Fig. 1-11) is taken out from pipe.Sample is ready for detection now.This sample of transferase 12 00 μ l is to empty test tube.This sample is detected by using following mensuration examination bar.If the detection of more than one inspection test-strips will be carried out to sample, multiple 200 μ l samples can be transferred to single empty test tube.Open the tank being equipped with and measuring examination bar.From tank, take out one measure examination bar and again build tank at once.Keep away the film surface that manual-free touches examination bar central authorities.Be placed in and dilute the pipe of samples by measuring examination bar containing 200 μ l, under the arrow points making mensuration try on bar (Fig. 1-12).Set the timer to 20 minutes, or record tries measuring the time (Fig. 1-13) that bar adds to sample.Within 20 minutes, read testing result (Fig. 1-14) later.Then examination bar, pipe and swab are abandoned in bio-hazard product waste canister (Fig. 1-15).
embodiment 3: determine the sensitivity of kit, specificity and accuracy
Measure the sensitivity of mouth cavity fluid detection, specificity and accuracy in the present embodiment.Carry out the external certificate test of mouth cavity fluid HIV side direction detection the anonymous HIV clinic (Thai Red Cross AnonymousHIV Clinic) of the Red Cross of Thailand of Bangkok, THA, on-test, in April, 2004, is completed in June, 2004.986 go to the anonymous HIV clinic of the Red Cross of Thailand and the current object not accepting antiretroviral therapy accept voluntary HIV antibody detect and consulting.Adopt the sequence detection of object carry out this research, do not know result in advance.In addition, 37 known positives and the object of antiretroviral therapy (ARV) also accept voluntary HIV antibody and detect and consulting.Give object chance to agree to voluntarily provide additional sample to be detected by these tests.
The reference method used in anonymous HIV clinic is the OrgenicsRapid HIV-1/-2 Blood Test for preliminary screening, Doublecheck
tMiI.Use Bio-RadGenScreen
tMhIV-1/2 Version 2 ELISA and/or Fujirebio Serodia
-HIV (HIV-1 only) Particle Agglutination Test confirms the reactive sample of this detection.
Sensitivity is expressed as the percentage of number positive gained that the number positive that detects with reference detects divided by quick mouth cavity fluid.Specificity is expressed as the percentage of negative number gained that the negative number that detects with reference detects divided by quick mouth cavity fluid.Accuracy is expressed as the percentage of the total number gained consistent divided by result between quick mouth cavity fluid detection with reference detection of object.Result sees following table.
Table 1. is HIV-1/2 mouth cavity fluid detection-Recombinant HIV-1/ synthesis HIV-2 peptide fast
| Number of subjects | The detection method adopted | Positive | Negative | Sensitivity (%) | Specificity (%) | Accuracy (%) |
| 986 examples not carry out ARV37 example carrying out ARV amount to 1023 | Quick mouth cavity fluid detection reference detects quick mouth cavity fluid detection reference and detects the detection of quick mouth cavity fluid detection reference | 355 355 35 37 390 392 | 631 631 2 0 633 631 | 100％ 94.6％ 99.5％ | 100％ - 100％ | 100％ 94.6 99.8％ |
Table 2. fast HIV-1/2 mouth cavity fluid detects-synthesizes HIV-1/ synthesis HIV-2 peptide
| Number of subjects | The detection method adopted | Positive | Negative | Sensitivity (%) | Specificity (%) | Accuracy (%) |
| 986 examples not carry out ARV37 example carrying out ARV amount to 1023 | Quick mouth cavity fluid detection reference detects quick mouth cavity fluid detection reference and detects the detection of quick mouth cavity fluid detection reference | 353 355 35 37 388 392 | 633 631 2 0 635 631 | 99.4％ 94.6％ 99.0％ | 100％ - 100％ | 100％ 94.6 99.6％ |
Sequence table
<110> Beijing Kelipute Biopharmaceutical Technology Co., Ltd.
<120> mouth cavity fluid fast immune chromatographic detects
<130>OP050003P
<160>5
<170>PatentIn Ver.2.1
<210>1
<211>39
<212>PRT
<213> artificial sequence
<220>
<223> artificial sequence description: biotinylated mono-lysine
Connexon HIV-1 (gp41 peptide)
<220>
<221>MOD_RES
<222>(1)
The biotinylated Lys of <223>Xaa=
<220>
<221> disulfide
<222>(20)..(26)
<400>1
Xaa Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu Gly
1 5 10 15
Ile Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro Trp
20 25 30
Asn Ala Ser Gly Lys Leu Ile
35
<210>2
<211>22
<212>PRT
<213> artificial sequence
<220>
<223> artificial sequence description: biotinylated
HIV-1gp120 peptide
<220>
<221>MOD_RES
<222>(1)
The biotinylated Lys of <223>Xaa=
<400>2
Xaa Thr Glu Pro Leu Gly Val Ala Pro Thr Lys Ala Lys Arg Arg Val
1 5 10 15
Val Gln Arg Glu Lys Arg
20
<210>3
<211>35
<212>PRT
<213> artificial sequence
<220>
<223> artificial sequence description: biotinylated mono-lysine
Connexon HIV-2 (gp36 peptide)
<220>
<221>MOD_RES
<222>(1)
The biotinylated Lys of <223>Xaa=
<220>
<221>MOD_RES
<222>(35)
<223>Xaa=silk amide
<220>
<221> disulfide
<222>(20)..(26)
<400>3
Xaa Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala Arg Leu Asn
1 5 10 15
Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Val Pro Trp Val
20 25 30
Asn Asp Xaa
35
<210>4
<211>40
<212>PRT
<213> artificial sequence
<220>
<223> artificial sequence description: biotinylated pair of lysine
Connexon HIV-1 (gp41 peptide)
<220>
<221>MOD_RES
<222>(1)
The biotinylated Lys of <223>Xaa=
<220>
<221> disulfide
<222>(21)..(27)
<400>4
Xaa Lys Ile Leu Ala Val Glu Arg Tyr Leu Lys Asp Gln Gln Leu Leu
1 5 10 15
GlyIle Trp Gly Cys Ser Gly Lys Leu Ile Cys Thr Thr Ala Val Pro
20 25 30
Trp Asn Ala Ser Gly Lys Leu Ile
35 40
<210>5
<211>37
<212>PRT
<213> artificial sequence
<220>
<223> artificial sequence description: biotinylated pair of lysine
Connexon HIV-2 (gp36 peptide)
<220>
<221>MOD_RES
<222>(1)
The biotinylated Lys of <223>Xaa=
<220>
<221>MOD_RES
<222>(37)
<223>Xaa=silk amide
<220>
<221> disulfide
<222>(21)..(27)
<400>5
Xaa Lys Val Thr Ala Ile Glu Lys Tyr Leu Gln Asp Gln Ala Arg Leu
1 5 10 15
Asn Ser Trp Gly Cys Ala Phe Arg Gln Val Cys His Thr Thr Val Pro
20 25 30
Trp Val Asn Asp Xaa
35
Claims (5)
1. lateral flow immunochromatography inspection test-strips is for the preparation of the purposes detected in the kit of HIV antibody in mouth cavity fluid,
Wherein said lateral flow immunochromatography inspection test-strips comprises sample pad, conjugate pad, detection zone and check plot pad, and described pad is made up of at least one host material, wherein
Described conjugate pad is positioned at the downstream of described sample pad, and draws striped thereon with conjugate;
Described detection zone and check plot pad are positioned at the downstream of conjugate pad, and containing detection zone and check plot, wherein
Described detection zone is fixed with the specificity combinating reagent of specific binding target analyte; And
Described check plot is fixed with the second capture agent; And
The wherein said host material for sample pad is glass fiber filter paper, and the described host material for conjugate pad is polyester material, and the described host material for detection zone and check plot pad is nitrocellulose filter; And
Wherein said conjugate comprises the label with the first capture agent coupling, described first capture agent catches the endogenous antibody of mouth cavity fluid, described label is colloid gold particle, and described first capture agent is selected from the antibody of anti-igg, IgM or IgA, albumin A, Protein G and concanavalin A; And
Wherein said specificity combinating reagent is the antigen of communicable disease, and the antigen of described communicable disease is the restructuring or the synthetic peptide that represent HIV protein immunization determining area; And
Wherein said second capture agent is selected from the antibody of anti-igg, IgM or IgA, albumin A, Protein G and concanavalin A.
2. purposes according to claim 1, wherein said HIV albumen is HIV envelope protein.
3. purposes according to claim 2, wherein said HIV envelope protein is selected from gpl20 and gp41 of HIV-1 and the gp36 of HIV-2.
4. purposes according to claim 1, wherein said anti-igg antibody is goat anti-human IgG antibodies.
5. purposes according to claim 1, wherein said first capture agent is albumin A.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2005/000701 WO2006122450A1 (en) | 2005-05-20 | 2005-05-20 | Oral fluid rapid immunochromatography test |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410158273.9A Division CN103983773A (en) | 2005-05-20 | 2005-05-20 | Fast immunochromatographic detection of oral fluid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101495865A CN101495865A (en) | 2009-07-29 |
| CN101495865B true CN101495865B (en) | 2015-01-28 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200580049809.2A Active CN101495865B (en) | 2005-05-20 | 2005-05-20 | Rapid immunity chromatography detection of mouth cavity fluid |
Country Status (6)
| Country | Link |
|---|---|
| US (1) | US20110003310A1 (en) |
| EP (1) | EP1882184A4 (en) |
| CN (1) | CN101495865B (en) |
| BR (1) | BRPI0520182A2 (en) |
| MX (1) | MX2007014476A (en) |
| WO (1) | WO2006122450A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070092978A1 (en) * | 2005-10-20 | 2007-04-26 | Ronald Mink | Target ligand detection |
| BR112012006757A2 (en) * | 2009-09-25 | 2015-09-08 | Alere San Diego Inc | nucleic acid detection in raw matrices |
| CN102192979A (en) * | 2010-03-08 | 2011-09-21 | 苏州浩欧博生物医药有限公司 | Lateral chromatographic one-step analytical method utilizing non-bibulous film, and assay kit |
| CN101943699B (en) * | 2010-08-31 | 2013-04-03 | 广州万孚生物技术股份有限公司 | Test strip for detecting HIV antibodies in spittle and preparation method thereof |
| CN102103145B (en) * | 2011-02-24 | 2013-01-16 | 南京基蛋生物科技有限公司 | Colloidal gold test strip for double-amplification system and preparation method thereof |
| CN102323255B (en) * | 2011-05-30 | 2013-08-14 | 吉权 | Ischemia modified albumin determination colour-developing agent and preparation method thereof |
| CN102288758A (en) * | 2011-07-29 | 2011-12-21 | 北京中检安泰诊断科技有限公司 | Hepatitis A virus (HAV) IgM antibody colloidal gold method detection kit and preparation method thereof |
| CN102565382B (en) * | 2012-01-06 | 2013-12-04 | 苏州浩欧博生物医药有限公司 | Immunochromatography method for detecting allergen-specific IgE antibodies in blood samples |
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- 2005-05-20 BR BRPI0520182-9A patent/BRPI0520182A2/en not_active IP Right Cessation
- 2005-05-20 US US11/920,747 patent/US20110003310A1/en not_active Abandoned
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|---|---|
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| MX2007014476A (en) | 2008-02-07 |
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| US20110003310A1 (en) | 2011-01-06 |
| CN101495865A (en) | 2009-07-29 |
| WO2006122450A1 (en) | 2006-11-23 |
| EP1882184A1 (en) | 2008-01-30 |
| EP1882184A4 (en) | 2008-07-30 |
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